Cold-induced expression vector

ABSTRACT

A vector having a region encoding a cold shock protein gene mRNA-origin 5′-nontranslated region, characterized in that the 5′-nontranslated region has a mutation having been transferred therein so as to change the distance of the stem structure formed by the region.

TECHNICAL FIELD

The present invention relates to a vector used for genetic engineeringand a method for expressing proteins using said vector.

BACKGROUND ART

Techniques for production of useful proteins using genetic engineeringare widely used nowadays. Among these, expression systems usingEscherichia coli as a host are the most commonly used expressionsystems. Many proteins have been produced using recombinants. Aso-called expression vector is generally constructed and used for theproduction of useful proteins using recombinants. In the expressionvector, a gene of interest is placed under the control of a promoterwhich is recognized by an RNA polymerase. Exemplary promoters used forexpression vectors for Escherichia coli as a host are lac, trp, tac, galand ara promoters. Expression vectors that utilize promoters other thanthose directly recognized by Escherichia coli RNA polymerase include thepET-system (Novagen). The pET-system utilizes a promoter recognized byan RNA polymerase from bacteriophage T7 which infects Escherichia coli(see J. Mol. Biol., 189:113-130 (1986); Gene, 56:125-135 (1987)). Incase of the pET-system, T7 RNA polymerase is expressed in Escherichiacoli, a gene of interest placed downstream of T7 promoter in anexpression vector is transcribed by T7 RNA polymerase, and the proteinof interest is synthesized using the translation system of the host.

However, if a protein of interest is expressed at a high level using oneof many Escherichia coli expression systems including the pET-system,the protein of interest may form an insoluble complex called inclusionbody in many cases. As a result, the amount of the protein of interestin its active form is greatly reduced. It has been reported for severalpolypeptides that active polypeptides were obtain by solubilization andrefolding of inclusion bodies. The recovery rates are generally low inmany cases. In addition, appropriate refolding conditions need to beexamined for each protein of interest. Thus, a system for directlyexpressing an active protein in Escherichia coli has been desired.

It is considered that inclusion bodies are formed as a result of thefollowing. An intermediate of a translated polypeptide chain prior tofolding into its proper conformation is interwound with anotherpolypeptide chain due to intermolecular interaction to form a hugeinsoluble complex. In such a case, it is known that the expression levelof a protein in its active form is increased by culturing recombinantEscherichia coli cells at a temperature lower than the conventional one37° C. (20 to 30° C.). It is supposed that this is because the slowtranslation by ribosome provides a sufficient time for the intermediateto be folded into its proper structure, and the slow action ofintracellular proteolytic enzyme under the low-temperature conditionsincreases the stability of expressed active protein. Thus, attention hasbeen paid to a method in which recombinant Escherichia coli cells arecultured under low-temperature conditions as being useful for producinga protein that forms inclusion bodies.

If a culture temperature for Escherichia coli cells during thelogarithmic growth phase is lowered from 37° C. to 10-20° C., growth ofthe Escherichia coli cells is temporarily arrested, during whichexpression of a group of proteins called cold shock proteins is induced.The proteins are classified based on the induction level into twogroups: a group I (10-fold or more) and a group II (less than 10-fold).Proteins in the group I include CspA, CspB, CspG and CsdA (see J.Bacteriol., 178:4919-4925 (1996); J. Bacteriol., 178:2994-2997 (1996)).Since the expression level of CspA (WO 90/09447) reaches 13% of thetotal cellular protein 1.5 hours after temperature shift from 37° C. to10° C. (see Proc. Natl. Acad. Sci. USA, 87:283-287 (1990)), attemptshave been made to utilize the promoter for the cspA gene for productionof a recombinant protein at a low temperature.

Regarding a system for expressing a recombinant protein underlow-temperature conditions using the cspA gene, the followingeffectiveness has been shown in addition to the above-mentioned highlyefficient transcription initiation by the promoter for the gene at a lowtemperature.

(1) If mRNA that is transcribed from the cspA gene and capable of beingtranslated does not encode a functional CspA protein (specifically, ifit encodes only a portion of the N-terminal sequence of the CspAprotein), such mRNA inhibits expression of other Escherichia coliproteins including cold shock proteins for a long period of time. Duringthis period, the mRNA is preferentially translated (J. Bacteriol.,178:4919-4925 (1996); WO 98/27220). This phenomenon is called LACE (lowtemperature-dependent antibiotic effect of truncated cspA expression)effect.

(2) A sequence consisting of 15 nucleotides called a downstream box islocated 12 nucleotides downstream of the initiation codon of the cspAgene. The translation efficiency under low-temperature conditions ismade high due to this sequence.

(3) A 5′-untranslated region consisting of 159 nucleotides is locatedbetween the transcription initiation site and the initiation codon inthe mRNA for the cspA gene. This region has a negative effect on theexpression of CspA at 37° C. and a positive effect under low-temperatureconditions.

In particular, the phenomenon as described in (1) above suggests thefeasibility of specific expression of only a protein of interestutilizing the cspA gene. Thus, it is expected that the system can beapplied to production of highly pure recombinant proteins or preparationof isotope-labeled proteins for structural analyses.

It is known that it may be difficult to culture an Escherichia coli cellcontaining an expression vector to a level at which the cell can besubjected to induction, or even construction of an expression vector maybe impossible if expression control of the promoter for the gene isincomplete and the gene product is harmful to the host (see, forexample, U.S. Pat. No. 5,654,169).

Modification of the expression vector has been tried using the5′-untranslated region of the cspA gene in order to solve theabove-mentioned problems, to further increase the gene expressionefficiency, and to readily obtain the expressed product (WO 99/27117).Modification by introducing an operator for making the expressioncontrol strict or by introducing a mutation into the 5′-untranslatedregion for increasing the gene expression level is disclosed therein.

DISCLOSURE OF INVENTION

As described above, the protein expression system under low-temperatureconditions is considered to be a very effective system, and furtherimprovements of expression efficiency and the like are desired.

The main object of the present invention is to construct a moreexcellent system for gene expression under low-temperature conditions,for example, by increasing gene expression efficiency of an expressionvector utilizing a csp gene.

In order to achieve this object, the present inventors have assessed theeffect of modification introduced into a nucleotide sequence of a5′-untranslated region derived from a csp gene. As a result, the presentinventors have found that adjustment of a distance between stems formedin a portion corresponding to the 5′-untranslated region of mRNAincreases the expression level of a protein encoded by the downstreamsequence. Furthermore, the present inventors have constructed a vectorcontaining a DNA that encodes such a modified 5′-untranslated region.Thus, the present invention has been completed.

The present invention is outlined as follows. The first aspect of thepresent invention relates to a vector having a portion encoding a5′-untranslated region derived from an mRNA for a cold shock proteingene, wherein a mutation is introduced into the 5′-untranslated regionsuch that a distance between stem structures formed in said region isaltered.

According to the first aspect, the mutation introduced into the5′-untranslated region is exemplified by insertion or deletion of anucleotide. A vector in which the mutation is introduced into a regionof the Escherichia coli cspA gene corresponding to nucleotide 593 tonucleotide 598 in SEQ ID NO:1 exemplifies a particularly preferableembodiment.

In the vector according to the first aspect, the portion encoding a5′-untranslated region may further have an operator. A 5′-untranslatedregion that has the nucleotide sequence of SEQ ID NO:2, 3 or 4 is aparticularly preferable example of the 5′-untranslated region in thevector.

The vector of the first aspect may have a promoter located upstream ofthe portion encoding a 5′-untranslated region. In addition, it may havea nucleotide sequence that is complementary to an anti-downstream boxsequence in a ribosormal RNA of a host to be used, wherein saidnucleotide sequence is located downstream of the portion encoding a5′-untranslated region.

For example, the vector of the first aspect may be a plasmid vector.

The second aspect of the present invention relates to a method forexpressing a protein of interest, the method comprising:

-   -   (1) transforming a host with the vector of the first aspect into        which a gene encoding a protein of interest has been        incorporated to obtain a transformant;    -   (2) culturing the transformant; and    -   (3) shifting the culture temperature down to one lower than a        conventional temperature to express the protein of interest.

According to the second aspect, a promoter may be induced during orafter step (3).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

As used herein, “a region” refers to an area of a nucleic acid (DNA orRNA). As used herein, “a 5′-untranslated region of an mRNA” refers to aregion that does not encode a protein, and is located on the 5′ side ofan mRNA synthesized as a result of transcription from a DNA.Hereinafter, the region may also be referred to as “a 5′-UTR(5′-Untranslated Region)”. Unless otherwise noted, the 5′-UTR refers tothe 5′-untranslated region of mRNA for the Escherichia coli cspA gene ora modification thereof.

As used herein, “a cold shock protein gene” refers to a gene encoding aprotein whose expression is induced upon lowering the growth temperatureof an organism from its physiological temperature.

A portion encoding a 5′-UTR of a cold shock protein mRNA used accordingto the present invention is a portion that encodes a region 5′ to theinitiation codon in the mRNA for the gene. Such a portion ischaracteristically found in an Escherichia coli cold shock protein gene(cspA, cspB, cspG, csdA or the like) (J. Bacteriol., 178:4919-4925(1996); J. Bacteriol., 178:2994-2997 (1996)). A portion of 100nucleotides or more from the 5′ end in the mRNA transcribed from such agene is not translated into a protein. This portion is important forcold response of gene expression. If this 5′-untranslated region isadded to an mRNA for an arbitrary protein at its 5′ end, translationfrom the mRNA into a protein takes place under low-temperatureconditions.

“A 5′-untranslated region of an mRNA” used according to the presentinvention is not limited to one derived from the above-mentionedspecific gene. A 5′-untranslated region that is derived from a coldshock protein gene and is capable of forming two or more stem structurescan be used according to the present invention.

Portions encoding 5′-UTRs derived from the cold shock protein genes aslisted above can be used for the vector of the present invention. Inparticular, one derived from the Escherichia coli cspA gene can bepreferably used. The nucleotide sequence of the Escherichia coli cspAgene is registered and available to the public under accession no.M30139 from the GenBank gene database. The nucleotide sequence is shownin SEQ ID NO:1. Furthermore, a 5′-UTR in which the nucleotide sequenceis partially modified can also be used according to the presentinvention. For example, one can use the 5′-UTR-encoding portion from thecspA gene contained in a plasmid pMM047 or pMM048 as described in WO99/27117. The nucleotide sequence of the 5′UTR-encoding portion from theEscherichia coli cspA gene contained in the plasmid pMM048 is shown inSEQ ID NO:5. This sequence is identical to the nucleotide sequence ofthe portion encoding the 5′-UTR contained in the plasmid pCold08NC2 usedin Examples in the present specification.

The present invention is characterized in that a mutation is introducedinto a 5′-UTR-encoding portion such that the mutation adjusts a distancebetween stems which are secondary structures presumably formed in the5′-UTR. For example, a distance between stems can be increased ordecreased by inserting nucleotide(s) into a portion corresponding to aregion between the stems or by deleting a part of nucleotides in such aportion. There is no specific limitation concerning the position atwhich a mutation is introduced, or the number of nucleotide(s) to beinserted or deleted in a mutation as long as the expression level of adownstream linked gene is elevated as a result of the mutation ascompared with one without the mutation. It is needless to say that themutation preferably does not interfere with the ability of the 5′-UTR toachieve cold-specific gene expression.

It is expected that the 5′-UTR derived from the Escherichia coli cspAgene forms stem structures in a region from nucleotide 584 to nucleotide593 and a region from nucleotide 598 to nucleotide 608 in the nucleotidesequence of SEQ ID NO:1. Thus, the distance between the stems can beadjusted by introducing insertion or deletion of nucleotide(s) into aportion between the above-mentioned regions (from nucleotide 593 tonucleotide 598) or a portion corresponding to said portion in a 5′-UTRhaving an introduced mutation. The present invention is not limited tothe above-mentioned adjustment of distance between stems.

The deletion mutations according to the present invention includedeletion of one to all of nucleotides in a portion between stems.Insertion mutations are exemplified by insertion of 1 to 100nucleotide(s), preferably 3 to 60 nucleotides, more preferably 8 to 40nucleotides.

Although it is not intended to limit the present invention, the 5′-UTRscontained in the plasmid vectors described in Examples, pCold08s2,pCold08s12 and pCold08s32, exemplify preferred embodiments of thepresent invention. The nucleotide sequences of the 5′-UTRs contained inthe plasmid vectors are shown in SEQ ID NOS:2, 3 and 4, respectively.

The expression vector of the present invention can be prepared using aportion encoding a 5′-UTR which is prepared according to the presentinvention and in which a distance between stems is adjusted as describedabove. Specifically, it may be prepared as follows. Secondary structuresformed by an mRNA encoded by a 5′-UTR-encoding DNA as a startingmaterial are assumed. An insertion or deletion mutation is introducedinto a portion presumably corresponding to a portion between stems usinga known method. The thus obtained DNA is incorporated into a vectoralong with an appropriate promoter.

The vector of the present invention may be any one of commonly usedvectors (e.g., plasmid, phage or virus vectors) as long as it can beused to achieve the object as a vector. It does not create inconvenienceif the vector of the present invention may be integrated into a genomicDNA in a host after transferred into the host.

The 5′-UTR is inserted between a promoter and a region encoding aprotein of interest in the vector of the present invention. Examples ofthe promoters used according to the present invention include, but arenot limited to, promoters derived from cold shock protein genes (e.g.,cspA, cspB, cspG and cspA) which are expected to have high promoteractivities at a low temperature. The promoter may be any one if it hasan activity of initiating transcription into RNA in a host to be used.If Escherichia coli is to be used as a host, a promoter such as the lac,trp, tac, gal or ara promoter can be used.

Regarding a region contained in addition to the above-mentionedelements, the vector of the present invention may have, for example, areplication origin, a drug-resistance gene used as a selectable marker,or a regulatory sequence such as an operator or a terminator.

Operators that are present in expression-regulatory regions of variousgenes such as the lac operator derived from the Escherichia coli lactoseoperon can be used according to the present invention. A promoter can beallowed to act by canceling the function of the lac operator using anappropriate inducer such as lactose or an analog thereof (preferably,isopropyl-β-D-thiogalactoside (IPTG)). Such an operator sequence isusually placed downstream of a promoter and near a transcriptioninitiation site.

The vector of the present invention may further have a regulatory genenecessary for a function of an operator (e.g., the lacI^(q) gene for thelac operator).

It is possible to increase expression efficiency by including,downstream of a 5′-untranslated region, a nucleotide sequencecomplementary to an anti-downstream box sequence in ribosomal RNA of ahost to be used, in addition to the above-mentioned elements. Forexample, in case of Escherichia coli, an anti-downstream box sequence ispresent from position 1467 to position 1481 in 16S ribosomal RNA (TheEMBO Journal, 15:665-674 (1996)). It is possible to use a regionencoding an N-terminal peptide of a cold shock protein which contains anucleotide sequence highly complementary to this sequence. It iseffective to place a sequence complementary to an anti-downstream boxsequence such that it starts from around the first to fifteenthnucleotide from the initiation codon. A gene encoding a protein ofinterest can be incorporated into a vector such that the protein isexpressed as a fusion protein with the N-terminal peptide.Alternatively, a base substitution may be introduced using a sitedirected mutagenesis method such that a gene encoding a protein ofinterest becomes complementary to an anti-downstream box sequence. If agene encoding a protein of interest is incorporated into a vector suchthat the protein of interest is expressed as a fusion protein, thepeptide may be of any length as long as the activity of the protein ofinterest is not abolished. The vector for expressing a fusion proteinmay be engineered, for example, at the joint site so that a protein ofinterest can be separated from the fusion protein using an appropriateprotease. Alternatively, it may be engineered so that the fusion proteinis expressed as a protein fused with a peptide that can be utilized forpurification or detection. Examples of peptides that can be utilized forpurification of expressed proteins include, but are not limited to,histidine tag (His-Tag) and glutathione-S-transferase (GST).

For example, a protein of interest is expressed using the vector of thepresent invention constructed as a plasmid as follows. A transformantfor expressing the protein of interest can be obtained by cloning a geneencoding the protein into the plasmid vector of the present inventionand transforming an appropriate host with the plasmid. The transformantspecifically expresses the protein of interest by lowering the culturetemperature. If the vector has an operator, the function of the operatormay be canceled using an appropriate means to induce a promoter.

If the vector of the present invention is used for protein expression, aprotein of interest is preferentially translated due to theabove-mentioned LACE effect. As a result, the content of the protein ofinterest in a culture is high as compared with conventional expressionof recombinant proteins and, therefore, it is possible to prepare theprotein of interest with high purity. An isotope-labeled protein can beprepared by expressing a protein of interest in the presence of anappropriate isotope. The thus obtained highly pure labeled protein issuitable for structural analysis using NMR. For example, a culture or alysate of a culture can be directly subjected to NMR analysis.

EXAMPLES

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Among the procedures described herein, basic procedures includingplasmid preparation and restriction enzyme digestion were carried outaccording to the methods as described in T. Maniatis et al. (eds.),Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring HarborLaboratory. Unless otherwise noted, Escherichia coli JM109 was used as ahost for the plasmid construction as described below, and LB medium (1%Tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0) containing 50 μg/ml ofampicillin or LB agar medium prepared by adding agar at a concentrationof 1.5% to the LB medium and solidifying the resulting mixture was used.

Referential Example Construction of Plasmid pCold08NC2

pCold08NC2 was constructed based on the description of WO 99/27117using, as a starting material, a plasmid pMM047 harbored in Escherichiacoli JM109/pMM047 (FERM BP-6523) (deposited on Oct. 31, 1997 (date oforiginal deposit) at International Patent Organism Depositary, NationalInstitute of Advanced Science and Technology, AIST Tsukuba Central 6,1-1, Higashi 1-Chome, Tsukuba, Ibaraki 305-8566, Japan). The plasmidpCold08NC2 has the following in this order from upstream to downstream:the lac promoter, a modified Escherichia coli cspA gene-derived 5′-UTRand a multiple cloning site. In addition, the plasmid has the lacI gene,a downstream box sequence that is completely complementary to ananti-downstream sequence in the Escherichia coli 16S ribosomal RNA, ahistidine tag consisting of six histidine residues, and a nucleotidesequence encoding an amino acid sequence recognized by factor Xa.

Example 1 Construction of Vector pCold08s2 and Examination of ProteinExpression Level

(1) Construction of Plasmid Vector pCold08s2

An insertion mutation of 20 nucleotides was introduced as follows intothe 5′-UTR-encoding portion in the plasmid pCold08NC2 from ReferentialExample 1.

A PCR was carried out using the plasmid pCold08NC2 as a template as wellas a synthetic primer Sp20F (SEQ ID NO:6) and a primer CSPterR (SEQ IDNO:7). A reaction mixture containing 50 ng of pCold08NC2, 5 μl of Ex Taqbuffer, 8 μl of dNTP mix, 5 pmol of the primer Sp20, 5 pmol of theprimer CSPterR, 0.5 μl of Takara Ex Taq (Takara Bio) and sterile waterto a total volume of 50 μl was subjected to a PCR (30 cycles of 94° C.for 1 minute (denaturation), 55° C. for 1 minute (primer annealing) and72° C. for 1 minute (synthesis reaction)). The whole PCR reactionmixture was subjected to electrophoresis on 3% (w/v) low melting pointagarose gel. An about 300-bp amplified DNA fragment was purified fromthe gel, suspended in 5 μl of sterile water and used as an amplifiedfragment SC.

A PCR was carried out under similar conditions using a synthetic primerSp20R (SEQ ID NO:8) and a primer NheF2 (SEQ ID NO:9) as well as pCold08as a template. The whole PCR reaction mixture was subjected toelectrophoresis on 3% (w/v) low melting point agarose gel. An about150-bp amplified DNA fragment was purified from the gel, suspended in 5μl of sterile water and used as an amplified fragment SN.

A mixture containing 1 μl each of the amplified fragments SC and SN, 5μl of Ex Taq buffer, 5 μl dNTP mix and sterile water to a total volumeof 50 μl was heated at 94° C. for 10 minutes, cooled to 37° C. over 60minutes and incubated at 37° C. for 15 minutes. 0.5 μl of Takara Ex Taqwas added thereto, and the mixture was heated at 72° C. for 3 minutes.

5 pmol each of the primers NheF2 and CSPterR, 5 μl of Ex Taq buffer, 5μl of dNTP mix and sterile water to a total volume of 100 μl were addedto the reaction mixture. The resulting reaction mixture was subjected toa PCR (30 cycles of 94° C. for 1 minute, 55° C. for 1 minute and 72° C.for 2 minutes). The whole PCR reaction mixture was subjected toelectrophoresis on 3% (w/v) low melting point agarose gel. An about400-bp amplified DNA fragment was purified from the gel, suspended in 5μl of sterile water and used as an amplified fragment Sp20-1.

Sp20-1 was doubly digested with restriction enzymes NheI and EcoRI (bothfrom Takara Bio). The resulting DNA fragment was separated byelectrophoresis on 1% low melting point agarose gel, and then extractedand purified. The purified DNA fragment and pCold08 digested with NheIand EcoRI were mixed and ligated together using DNA Ligation Kit (TakaraBio). The ligation reaction mixture was used to transform Escherichiacoli JM109 and the transformants were grown on LB agar media containingampicillin. Plasmids were prepared from the resulting colonies, andsubjected to DNA sequencing. A plasmid into which the PCR product hadbeen properly inserted was selected and designated as pCold08s2. InpCold08s2, a nucleotide sequence 5′-GAGCGGATAACAATTTCACA-3′ (SEQ IDNO:11) is inserted between +120 and +121 in the 5′-UTR of pCold08NC2(SEQ ID NO:5). The transcription initiation site in the lac operator isdefined as +1. This position corresponds to the position betweennucleotide 597 and nucleotide 598 of the nucleotide sequence of the cspAgene as shown in SEQ ID NO:1. The nucleotide sequence of the 5′-UTRcontained in pCold08s2 is shown in SEQ ID NO:2.

(2) Construction of Plasmids for Expressing Proteins

(2)-1 Construction of Plasmid Vector pCold08s2-GFP

A gene encoding a GFP protein derived from Aequorea victoria wasincorporated into pCold082 as follows. A PCR was carried out using aplasmid pQBI63 (Takara Bio) as a template as well as a synthetic primerGFP-F (SEQ ID NO:11) and a primer GFP-R (SEQ ID NO:12). A reactionmixture containing 50 ng of pQBI63, 5 μl of Ex Taq buffer, 8 μl of dNTPmix, 5 pmol each of the primers GFP-F and GFP-R, 0.5 μl of Takara Ex Taq(Takara Bio) and sterile water to a total volume of 50 μl was subjectedto a PCR (30 cycles of 94° C. for 1 minute, 55° C. for 1 minute and 72°C. for 1 minute). The whole PCR reaction mixture was subjected toelectrophoresis on 3% (w/v) low melting point agarose gel. An about700-bp amplified DNA fragment containing the GFP gene was purified fromthe gel and suspended in 5 μl of sterile water. The DNA fragment wasdoubly digested with EcoRI and XbaI (Takara Bio). The digested DNAfragment was separated by electrophoresis on 1% low melting pointagarose gel, and then extracted and purified. The purified DNA fragmentand pCold08s2 digested with EcoRI and XbaI were mixed and ligatedtogether using DNA Ligation Kit (Takara Bio). The ligation reactionmixture was used to transform Escherichia coli JM109 and thetransformants were grown on LB agar media containing ampicillin.Plasmids were prepared from the resulting colonies, and subjected to DNAsequencing. A plasmid into which the PCR product had been properlyinserted was selected and designated as pCold08s2-GFP. In addition, theamplified DNA fragment containing the GFP gene was incorporated intopCold08NC2, which does not have an inserted sequence in the 5′-UTR, in asimilar manner to obtain pCold08-GFP.

(2)-2 Construction of Plasmid Vector pCold08s2-H296

A gene encoding the H296 fragment protein, which is a heparin-bindingpolypeptide derived from fibronectin, was incorporated into pCold082 asfollows.

A PCR was carried out using a plasmid pCH102 (U.S. Pat. No. 5,198,423)as a template as well as a synthetic primer H296-F (SEQ ID NO:13) and aprimer H296-R (SEQ ID NO:14). A reaction mixture containing 50 ng ofpCH102, 5 μl of Ex Taq buffer, 8 μl of dNTP mix, 5 pmol each of theprimers H296-F and H296-R, 0.5 μl of Takara Ex Taq and sterile water toa total volume of 50 μl was subjected to a PCR (30 cycles of 94° C. for1 minute, 55° C. for 1 minute and 72° C. for 1 minute). The whole PCRreaction mixture was subjected to electrophoresis on 3% (w/v) lowmelting point agarose gel. An about 1-kbp amplified DNA fragment waspurified from the gel and suspended in 5 μl of sterile water. The DNAfragment was doubly digested with EcoRI and BamHI (Takara Bio). Thedigested DNA fragment was separated by electrophoresis on 1% low meltingpoint agarose gel, and then extracted and purified. The purified DNAfragment containing the H296 gene and pCold08s2 digested with EcoRI andBamHI were mixed and ligated together using DNA Ligation Kit (TakaraBio). The ligation reaction mixture was used to transform Escherichiacoli JM109 and the transformants were grown on LB agar media containingampicillin. Plasmids were prepared from the resulting colonies, andsubjected to DNA sequencing. A plasmid into which the PCR product hadbeen properly inserted was selected and designated as pCold08s2-H296. Inaddition, the amplified DNA fragment containing the H296 gene wasincorporated into pCold08NC2, which does not have an inserted sequencein the 5′-UTR, in a similar manner to obtain pCold08-H296.

(2)-3 Construction of Plasmid Vector pCold08s2-lac

A plasmid pKM005 which contains the β-galactosidase (lacZ) gene (M.Inouye (ed.), Experimental Manipulation of Gene Expression, pp.15-32,1983, New York Academic Press) was digested with BamHI and SalI (TakaraBio). The reaction mixture was subjected to electrophoresis on 1% (w/v)low melting point agarose gel. A DNA fragment containing the lacZ genewas purified from the gel and suspended in 5 μl of sterile water. Thepurified DNA fragment and pCold08s2 digested with BamHI and SalI weremixed and ligated together using DNA Ligation Kit (Takara Bio). 10 μl ofthe ligation reaction mixture was used to transform Escherichia coliJM109, and the transformants were grown on LB agar media containingampicillin. Plasmids were prepared from the resulting colonies, andsubjected to DNA sequencing. A plasmid into which the PCR product hadbeen properly inserted was selected and designated as pCold08s2-lac. Inaddition, the amplified DNA fragment containing the lacZ gene wasincorporated into pCold08NC2, which does not have an inserted sequencein the 5′-UTR, in a similar manner to obtain pCold08-lac.

(3) Assessment of Expression Levels

(3)-1 Assessment of Expression Levels Using pCold08s2-GFP andpCold08s2-H296

Escherichia coli BL21 (Novagen) or CL83 (J. Bacteriol., 180:90-95(1998)) was transformed with pCold08s2-GFP (prepared in Example 1-(2)-1)or pCold08s2-H296 (prepared in Example 1-(2)-2), or pCold08-GFP orpCold08-H296 as a control. Each transformant was inoculated into 2.5 mlof LB medium containing 50 μg/ml of ampicillin and cultured overnight at37° C. with shaking. The culture was inoculated into 3 ml of the samemedium at a concentration of 1% (v/v) and cultured at 37° C. withshaking. When the turbidity (OD600 nm) reached 0.2, the culturetemperature was lowered to 15° C., and incubation was continued at thetemperature for 15 minutes. Then, IPTG was added thereto at a finalconcentration of 1 mM, and cultivation with shaking was continued for 24hours while maintaining the culture temperature at 15° C. Aftermeasuring the turbidity (OD600nm), cells collected from 2 ml of theculture by centrifugation were suspended in 100 μl of a cell suspensionsolution (50 mM tris-hydrochloride buffer (pH 7.5), 150 mM sodiumchloride). The suspension corresponding to about 3.75×10⁶ cells(calculated based on the turbidity) was subjected to electrophoresis on10% SDS polyacrylamide gel. The gel was stained with Coomassie BrilliantBlue (CBB) and then decolorized. SDS polyacrylamide gel electrophoresiswas carried out as described in “Seibutsukagaku Jikken No Tebiki 2”(Kagakudojin). The gel was subjected to image analysis using Total Labver.1.11 (Nonlinear Dynamics) to quantify the expression levels of GFPand H296. Ratios of the determined protein expression levels forrecombinants harboring pCold08s2-GFP and pCold08s2-H296 to those forrecombinants harboring pCold08-GFP and pCold08-H296, which do not haveinsertion in the 5′-UTRs, are shown in Table 1. TABLE 1 Plasmid HostExpression rate* pCold08s2-GFP BL21 1.5-fold CL83 2.0-foldpCold08s2-H296 BL21 1.5-fold CL83 1.5-fold*Expression rate: expression level for pCold08s2 clone/expression levelfor pCold08 clone, calculated based on results of image analyses.

Based on the results of SDS polyacrylamide gel analyses as shown inTable 1, expression levels for the clones prepared using the vectorpCold08s2, which has insertion of 20 nucleotides in the 5′-UTR, werehigher than those for the clones prepared using pCold08NC2.

(3)-2 Assessment of Expression Level Using pCold08s2-lac

Escherichia coli BL21 or CL83 was transformed with pCold08s2-lac(prepared in Example 1-(2)-3), or pCold08-lac as a control. Eachtransformant was inoculated into 2.5 ml of LB medium containing 50 μg/mlof ampicillin and cultured overnight at 37° C. with shaking. The culturewas inoculated into 3 ml of the same medium at a concentration of 1%(v/v) and cultured at 37° C. with shaking. When the turbidity (OD600 nm)reached 0.2, a sample was taken from the culture, the culturetemperature was lowered to 15° C., and incubation was continued at thetemperature for 15 minutes. Then, IPTG was added thereto at a finalconcentration of 1 mM to induce the expression, and cultivation wascontinued while maintaining the culture temperature at 15° C. Samplestaken from the culture at 37° C. just before the induction and theculture 3 or 24 hours after the induction were subjected toβ-galactosidase activity measurements according to the method asdescribed in J. H. Miller, Experiments in Molecular Genetics,pp.352-355, 1972, Cold Spring Harbor Laboratory. The results are shownin Table 2. TABLE 2 β-Galactosidase activity (unit) 3 hours 24 hoursBefore after after Plasmid Host induction induction inductionpCold08-lac BL21 10110  49510 85550 CL83 6971 28514 31504 pCold08s2-lacBL21 9151 66314 119770  CL83 5172 56028 72459

As shown in Table 2, the transformant harboring pCold08s2-lac, which hasinsertion of 20 nucleotides in the 5′-UTR, had a β-galactosidaseactivity higher than the transformant harboring pCold08-lac (1.4-fold(host: BL21) or 2.3-fold (host: CL83)) 24 hours after the induction.

Example 2 Construction of Vectors pCold08s12 and pCold08s32 andExamination of Protein Expression Levels

(1) Construction of Plasmid Vector pCold08s12

A plasmid pCold08s12 was constructed as described in Example 1-(1)except that primers Sp12F (SEQ ID NO:15) and Sp12R (SEQ ID NO:16) wereused in place of the primers Sp20F and CSPterR. In the plasmidpCold08s12, an insertion mutation of 12 nucleotides is introduced in the5′-UTR-encoding portion in the plasmid pCold08NC2 from ReferentialExample 1.

In pCold08s12, a nucleotide sequence 5′-ATGTTTTGTAGA-3′ (SEQ ID NO:17)is inserted between +120 and +121 of the 5′-UTR in pCold08NC2 (SEQ IDNO:5). The nucleotide sequence of the 5-UTR contained in pCold08s12 isshown in SEQ ID NO:3.

(2) Construction of Plasmid Vector pCold08s32

A plasmid pCold08s32 was constructed as described in Example 1-(1)except that primers Sp32F (SEQ ID NO:18) and Sp32R (SEQ ID NO:19) wereused in place of the primers Sp20F and Sp20R. In the plasmid pCold08s32,an insertion mutation of 32 nucleotides is introduced in the5′-UTR-encoding portion in the plasmid pCold08NC2 from ReferentialExample 1.

In pCold08s32, a nucleotide sequence5′-ATGTTTTGTAGATTTGAAAGAGTAGATTAGTA-3′ (SEQ ID NO:20) is insertedbetween +120 and +121 of the 5′-UTR in pCold08NC2 (SEQ ID NO:5). Thenucleotide sequence of the 5′-UTR contained in pCold08s32 is shown inSEQ ID NO:5.

(3) Construction of Plasmid Vectors pCold08s12-H296 and pCold08s32-H296

An about 1-kbp DNA fragment containing the H296 gene prepared asdescribed in Example 1-(2)-1 was doubly digested with EcoRI and BamHI.The digested DNA fragment was separated by electrophoresis on 1% lowmelting point agarose gel, and then extracted and purified. The purifiedDNA fragment and pCold08s12 or pCold08s32 digested with EcoRI and BamHIwere mixed and ligated together using DNA Ligation Kit (Takara Bio). Theligation reaction mixture was used to transform Escherichia coli JM109,and the transformants were grown on LB agar media containing ampicillin.Plasmids were prepared from the resulting colonies, and subjected to DNAsequencing. Plasmids into which the PCR product had been properlyinserted were selected and designated as pCold08s12-H296 andpCold08s32-H296.

(4) Assessment of Expression Levels Using pCold08s12-H296 andpCold08s32-H296

Escherichia coli BL21 or CL83 was transformed with pCold08s12-H296 orpCold08s32-H296 prepared in (3) above, or pCold08-H296 as a control.Each transformant was inoculated into 2.5 ml of LB medium containing 50μg/ml of ampicillin and cultured overnight at 37° C. with shaking. Theculture was inoculated into 3 ml of the same medium at a concentrationof 1% (v/v) and cultured at 37° C. with shaking. When the turbidity(OD600 nm) reached 0.2, the culture temperature was lowered to 15° C.,and incubation was continued at the temperature for 15 minutes. Then,IPTG was added thereto at a final concentration of 1 mM, and cultivationwith shaking was continued for 24 hours while maintaining the culturetemperature at 15° C. The expression levels of H296 for the respectiverecombinants were quantified for the resulting cultures as described inExample 1-(3)-1. Ratios of the determined protein expression levels forrecombinants harboring pCold08s12-H296 and pCold08s32-H296 to that for arecombinant harboring pCold08-H296, which does not have insertion in the5′-UTR, are shown in Table 3. TABLE 3 Plasmid Host Expression rate*pCold08s12-H296 BL21 1.3-fold CL83 1.5-fold pCold08s32-H296 BL211.4-fold CL83 1.8-fold*Expression rate: expression level for pCold08s12 or pColds32clone/expression level for pCold08 clone, calculated based on results ofimage analyses.

The results of SDS polyacrylamide gel analyses proved that theexpression levels observed using the vectors having the 5′-UTR withinserted 12 or 32 nucleotides were increased as compared with theexpression level observed using the vector without the insertion.

Industrial Applicability

The present invention provides an expression vector that results in highexpression efficiency under low-temperature conditions. It is possibleto specifically express a protein of interest to prepare a highly pureprotein preparation using the vector. In addition, it is possible toefficiently obtain a protein retaining its activity by expressing theprotein under low-temperature conditions utilizing the vector.

Sequence Listing Free Text

SEQ ID NO:2: Modified 5′ Untranslated Region of cspA Gene

SEQ ID NO:3: Modified 5′ Untranslated Region of cspA Gene

SEQ ID NO:4: Modified 5′ Untranslated Region of cspA Gene

SEQ ID NO:5: Modified 5′ Untranslated Region of cspA Gene

SEQ ID NO:6: Synthetic Primer for PCR

SEQ ID NO:7: Synthetic Primer for PCR

SEQ ID NO:8: Synthetic Primer for PCR

SEQ ID NO:9: Synthetic Primer for PCR

SEQ ID NO:10: Synthetic Nucleotide inserted into 5′ Untranslated Regionof cspA Gene

SEQ ID NO:11: Synthetic Primer for PCR

SEQ ID NO:12: Synthetic Primer for PCR

SEQ ID NO:13: Synthetic Primer for PCR

SEQ ID NO:14: Synthetic Primer for PCR

SEQ ID NO:15: Synthetic Primer for PCR

SEQ ID NO:16: Synthetic Primer for PCR

SEQ ID NO:17: Synthetic Nucleotide inserted into 5′ Untranslated Regionof cspA Gene

SEQ ID NO:18: Synthetic Primer for PCR

SEQ ID NO:19: Synthetic Primer for PCR

SEQ ID NO:20: Synthetic Nucleotide inserted into 5′ Untranslated Regionof cspA Gene

1. A vector having a portion encoding a 5′-untranslated region derivedfrom an mRNA for a cold shock protein gene, wherein a mutation isintroduced into the 5′-untranslated region such that a distance betweenstem structures formed in said region is altered.
 2. The vectoraccording to claim 1, wherein the introduced mutation is insertion ordeletion of a nucleotide.
 3. The vector according to claim 1, whereinthe mutation is introduced into a region corresponding to nucleotide 593to nucleotide 598 in SEQ ID NO:1.
 4. The vector according to claim 1,wherein the portion encoding a 5′-untranslated region further has anoperator.
 5. The vector according to claim 4, wherein the portionencoding a 5′-untranslated region is a portion encoding a5′-untranslated region that has the nucleotide sequence of SEQ ID NO:2,3 or
 4. 6. The vector according to claim 1, which has a promoter locatedupstream of the portion encoding a 5′-untranslated region.
 7. The vectoraccording to claim 1, which has a nucleotide sequence that iscomplementary to an anti-downstream box sequence in a ribosormal RNA ofa host to be used, wherein said nucleotide sequence is locateddownstream of the portion encoding a 5′-untranslated region.
 8. Thevector according to claim 1, which is a plasmid vector.
 9. A method forexpressing a protein of interest, the method comprising: (1)transforming a host with the vector defined by claim 1 into which a geneencoding a protein of interest has been incorporated to obtain atransformant; (2) culturing the transformant; and (3) shifting theculture temperature down to one lower than a conventional temperature toexpress the protein of interest.
 10. The method according to claim 9,wherein a promoter is induced during or after step (3).